The long-term goal of our research is to understand the structural basis for selective permeation of water and polyols through human aquaporins and aquaglyceroporins. This proposed research will explore how selective permeability is built into these transmembrane channels, how substrate permeation actually occurs at a chemical level and how certain residues confer selective permeability upon human isoforms. Toward these ends, we will use X-ray crystallography to determine structures of two homologous bacterial isoforms in complex with their substrates. We will correlate structure and sequence to identify residues that determine selective permeability. Mutagenesis will then be used to introduce human residues into the bacterial templates. Protein expression, purification and reconstitution will be used to generate human-like bacterial variants, which will be incorporated into proteoliposomes, where selective permeability will be examined by stopped-flow measurements. Aim (l) will determine the structure of GlpF-water, an E. coli aquaglyceroporin bound with water molecules. Comparing structures of GlpF-water and GlpF-glycerol will elucidate how a glycerol molecule displaces water while passing through the transmembrane channel. Aim (2) will determine the structures of GlpF in complex with ribitol and its stereoisomer xylitol to elucidate the structural basis for stereo-selectivity in GlpF. Aim (3) will crystallize AqpZ, an E. coli aquaporin, and determine its structure. Comparing structures of AgpZ and GlpF will reveal residues that determine selective permeation of water over glycerol. Aim (4) will investigate how determinants of water and glycerol permeation confer channel selective permeability upon human isoforms. Human determinants will be swapped into AqpZ or GlpF. The selective permeability of human determinants will be characterized in bacterial templates and evaluated by comparing with that of cognate human isoforms. Human aquaporins and aquaglyceroporins regulate osmotic balance of water and small non-electrolytes in health and disease. Completion of this proposed study will set the stage for structure-based drug design targeting human osmoregulation disorders, while the general findings will elucidate the mechanism of selective permeability through a crystallized membrane channel at a chemical level.